ANTIFUNGAL PROPERTIES OF VARIOUS EXTRACTS OF BACILLUS SUBTILIS (TRACY STRAIN) OBTAINED IN THE BACITRACIN RECOVERY PROCESS*

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1 ANTIFUNGAL PROPERTIES OF VARIOUS EXTRACTS OF BACILLUS SUBTILIS (TRACY STRAIN) OBTAINED IN THE BACITRACIN RECOVERY PROCESS* MORRIS MOORE, PH.D.f AND WILFRED E. WOOLDRIDGE, M.D4 It has been known for some time that bacteria, either spore-formers or non-spore-formers are able to inhibit or alter the growth of a variety of bacteria and fungi. Many of the early papers on this subject have been reviewed by Waksman in a paper published in 1941 (1). Many other observations on antibiosis have never been published. The antibiotic activity of these bacteria was directed towards or observed on fungi which were either plant pathogens or saprophytes. In a report published in 1927, Weidman (2) remarked that he had noted several years previously that "portions of a certain ringworm fungus colony which were adjacent to a bacterial colony did not develop as luxuriantly as the more remote parts". The following year, Chambers and Weidman (3) while searching for organisms with "inhibitory properties against fungi" reported isolating bacteria from the normal toe webs of four patients. These bacteria were identified as Bacillus subtilis. They mixed bacterial colonies with 11 separate species of fungi. When the mixtures were cultured, they found that the bacteria completely inhibited the growth of the fungi. In their clinical work, they applied living organisms of Bacillus subtilis to toes affected with fungi and obtained up to 50 per cent improvement. While the senior author was working in in the mycological laboratory of Washington University, he noted that the stock cultures of pathogenic fungi planted on mediums made with shredded agar became overrun with a bacterium identified as Bacillus subtilis. The fungi were completely inhibited in growth and this was attributed to the bacterium which was later isolated in large numbers from the contaminated agar-agar. Tests made at a later date showed that this spore-forming bacillus was more resistant to sterilization than the usual, contaminating non-spore-forming bacteria. As to the finding of Bacillus subtilis in cultures for fungi, we have noted that when the bacterium is present in the skin scrapings, the growth of pathogenic fungi is invariably inhibited in the isolation medium. Bacillus subtilis is a fairly common contaminant of lesions of the toes, fingers and nails. In 1942, Katznelson (4) obtained a substance from an aerobic spore-forming bacillus which inhibited the growth of 71 out of 81 species of parasitic and saprophytic fungi, including also some gram-positive bacteria. The toxic substance was thermostabile and diffusible. In 1946, Lewis, Hopper and Shultz (5) reported the chance contamination of an experimental culture plate seeded with spores of Trichophyton mentagrophytes CI'. gypseum) and with Bacillus subtilis to determine the inhibiting action of the bacterium on the fungus. The inoculated B. subtilis had no effect on the fungus. The contaminant, however, later * From the Department of Dermatology of the Barnard Free Skin and Cancer Hospital and the School of Medicine, Washington University, St. Louis, Mo. Service of Richard S. Weiss, M.D. f Mycologist to the Barnard Free Skin and Cancer Hospital; the Barnes Hospital; and the Department of Dermatology, School of Medicine, Washington University. Research Assistant, Department of Dermatology, School of Medicine, Washington University and Assistant Dermatologist, the Barnard Free Skin and Cancer Hospital. This study was supported by a grant from the Commercial Solvents Corporation, New York City. Received July 27,

2 266 THE TOURNAL OF INVESTIGATIVE DERMATOLOGY identified as a variant of B. subtilis and named var. XG and XY, produced a definite zone of inhibition. A Seitz ifitrate of the bacterium contained the inhibitory principle which remained potent after 11 months storage on ice. These experiments with cultures of the living bacterium, Bacillus subtilis, and with the crude filtrates, demonstrated an inhibitory substance which was definitely able to arrest the growth of fungi as well as various species of gram-positive and gram-negative bacteria. With the development of the antibiotic penicillin in crystalline form and with the demonstration of its therapeutic possibilities, there was a sudden surge in an attempt to uncover new antibiotics. These new therapeutic agents were evolved from numerous fungi and bacteria and the list is growing steadily and rapidly. One of the most fruitful organisms for the production of different antibiotics is Bacillus subtilis. More inhibitory substances have been produced by this organism on a variety of mediums both inorganic and organic, with or without carbohydrates, than by any other known organism. Benedict and Langlykke (6) list five and possibly six antibiotics which have been crystallized. At the head of the list is subtitin, first described by Jansen and Hirschmann (7). This substance is active chiefly against gram-positive bacteria, mycobacteria and some pathogenic fungi (8). A second antibiotic, bacitracin, first described by Johnson, Anker and Meleney (9) was isolated from a strain ("Tracy 1") of Bacillus subtilis. It resembles subtilin in many ways but differs from it in that bacitracin is formed in the shallow layers of the medium, that is in the culture medium free of the cells and not in the submerged growth. Bacitracin is somewhat toxic but non-irritating to tissue, neutral, soluble in water and lower alcohols, relatively heat stable and resists digestion with trypsin or pepsin. It is active against grampositive organisms, gonococcus, meningococcus and some actinomycetes. Bacillin, a third antibiotic (anticoli factor) was reported by Foster and Woodruff (10). Carbohydrate appears to be essential in the medium for its production. It is highly active against gram-positive and gram-negative bacteria and is moderately toxic for mice. The antibacterial property of this antibiotic is reduced or eliminated when blood or other complex natural materials are present in the medium. Another antibiotic, eumycin, developed by the Marburg strain of Bacillus subtilis was reported by Johnson and Burdon (11). It is effective chiefly against Corynebacterium diphtheriae, Mycobacterium tuberculosis and some pathogenic fungi. It is ineffective against gram-negative bacteria and weakly effective against staphylococci. Strongly fungistatic fractions of eumycin are hemolytic and toxic for mice. The French workers have reported other antibiotics produced by Bacillus subtilis. These include subtilysin (subtilyne) reported by Vallée (12) which is effective against some gramnegative and gram-positive bacteria; endosubtilysin of Saint-Rat and Olivier (13) which was reported as nontoxie and effective in high dilution against staphylococci; and a third antibiotic named subtiline by Ramon and Richou (14). An antibiotic named licheniformin by Callow and D'Arcy Hart (15) and obtained from Bacillus licheniformis is considered to he identical with subtilin by Benedict and Langlykke with the organism probably identical with the Ford strain of Bacillus subtilis. In 1947, Landy, Rosenman and Warren (16) reported briefly on an antibiotic developed by Bacillus subtilis which had negligible antibacterial activity but was strongly effective against pathogenic fungi. The antibiotic was produced in shallow layers ins to 6 days, was heat stable, not inactivated in a hydrogen ion range of 2.5 to 9.0, non-dialyzable through cellophane membranes and was not inactivated by body fluids. In a later publication in 1948, Landy, Warren, Rosenman and Colio (17) named the antibiotic bacillomycin because of its high antifungal and low antibacterial activity. In 1948, also, Hirschhorn, Bucca and Thayer (18) reported an additional antibiotic from Bacillus subtilis which the authors named subtenolin because of its "strong enolic properties". Highest yields of the antibiotic were obtained in a medium containing dl-alanine, manganese and copper. Media containing carbohydrate, peptones and other complex organic enriching substances were poor producers of subtenolin. Subtenolin was active

3 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 267 against staphylococci, certain gram-positive and gram-negative bacteria and partially active against Mycobacterium tuberculosis and is toxic for mice inoculated peritoneally, the LD,0 being 30 to 60 mgm. (30,000 to 60,000 units). A study of the chemical properties of subtenolin by Howell and Tauber (19) showed that subtenolin differed chemically from streptomycin, penicillin G, gramicidin and bacitracin. It is apparent that Bacillus subtilis in crude form live whole cultures and the various antibiotics which it produces have a varying degree of antifungal activity. In a search for an agent which would be strongly effective against pathogenic fungi, it was felt that an attempt should be made to obtain, in pure form, the antifungal property from Bacillus subtilis. Of the known antibiotics, at the time the study was undertaken, bacitracin was not only made available in sufficient quantity but the various fractions used in the study were also prepared through the courtesy of the Commercial Solvents Corporation. This paper, therefore, is devoted to a study of the antifungal properties of Bacillus subtilis, of bacitracin, an antibiotic produced by the organism and of the various fractions derived during and after the process involved in the extraction of bacitracin. MATERIALS AND TECHNIC In the evaluation of the antifungal properties of Bacillus subtilis, cultures of living bacilli as well as various extracts obtained from the organism and from the medium in which the organism was grown were used. The strains of Bacillus subtilis employed were the Tracy I strain which yields the antibiotic bacitracin and a strain obtained from the Western Regional Research Laboratories, culture number 370, which produces the antibiotic subtilin. A third strain obtained from the Venereal Disease Research Laboratory at Staten Island, New York, culture number 370, apparently was identical with the strain from the Western Regional Research Laboratory. In experiments with the live organisms, plates seeded with the various pathogenic fungi were inoculated at several points in the plates to determine the inhibitory effect of the bacillus on the fungus. Sabouraud's glucose agar or broth (Difco) was used throughout this work. A number of filtrates were also employed. These include a crude broth filtrate containing live cells, the lyophilized crude broth filtrate which was cell free and several filtrates obtained prior to and following the recovery of bacitracin. These may be best demonstrated in the accompanying schematic fiowsheet indicating the processes involved in the recovery of bacitracin. The organisms used for investigation in this paper were selected because of their common occurrence (producers of superficial dermatomycoses and of the invasive or systemic mycoses) and because of their ability to grow well. Unfortunately, in most instances, the amount of material available for use was insufficient in quantity, due to production difficulties, to warrant the use of a large number of different species of pathogenic fungi. Because of these circumstances, it was felt that the proper selection of a sufficiently representative number of species of fungi, well controlled, although small in number, would

4 268 'E JOURNAL OF INVESTIGATIVE DERMATOLOGY best evaluate the extract or fraction tested. To avoid needless repetition, the fungi used are listed in the tables, with the results. Whole culture experiments. In this series, plates seeded with suspensions of various fungi were inoculated with the strains of Bacillus subtilis. All cultures of fungi were, with the exception of Histoplasma capsulatum, nine days old. H. capsulatum was 26 days old. The cultures of B. subtilis were three to four days old. The inoculated plates were incubated at room temperature, approximately 25 C. and the final readings and photographs were made 17 days after inoculation (Fig. 1). This is a crude but significant form of experiment since it gives some clue as to the susceptibility of the fungi to the antifungal principle which is elaborated by the bacterium in its growth processes. To be sure, we are not dealing with Whole Collate (Breillos Sebtillo) Satv.nt A Grad. Broth Filtrate Ole. Cello) LyophiIled Grade Broth Filtrate Cell Free) Sport Boor Filtrate B l.oyer Spool Solvent A Filtrate C flltrote E I_Solvent Loper Spent Water Layer FiltrateD Loy.r Water l.ayer (Bocltroeltt) Filtrate F SCHEMATIC FLOWSNEET OF FII..TRATE RECOVERY a purified agent but a complex substance made up of several impure reactors. Consequently, it was hoped that should there be any inhibition of the fungi with the living organisms then better results should be expected with purified principles. Plates observed 17 days after inoculation revealed that in most instances Bacillus subtilis had grown rather widely due perhaps to the fact that the medium was fresh and that there was considerable moisture on the agar surface. The results, however, could be interpreted without difficulty. The yeastlike organisms, Cryptococcus neoformans, Monilia (Candida, Syringospora) albicans (Fig. 1, 4) and Sporotrichum schenckii were not affected by the bacterium, showing an unrestrained growth and in some plates overgrowth of the bacterial colonies. The dermatophytes, however, including Microsporum canis (Fig. 1, 1) M. gypseum (M. fulvum), Trichophyton mentagrophytes (Fig. 1, 2), Achorion

5 PROPERTIES OF EXTRACTS OF BACiLLUS SUBTILIS 269 Fin inoculated at three poiots with Bacillus subtilis and photographed 17 days after inoculation; 5 6 inoculated at one point with B. suhtilis aod photographed 9 days after inoculation. All grown on Sabouraud's (Difco) glucose agar. I. Microsporum canis; 2. Trichophton mentagrophytes; 3. Coccidioides immitis; 4. Monilia (Candida) albicans; 5. Nocardia asteroides; 6. N. brasiliensis. schoenleinii and Epidermophyton fioccosum exhibited definite zones of inhibition. This inhibitory effect Could also be clearly demonstrated with some of the fungi causing systemic mycoses. The chief systemic invaders affected were

6 270 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY Coccidioides immitis (Fig. 1, 3) and the aerobic actinomycetes, Nocardia asteroides (Fig. 1, 5), N. madurae and N. brasiliensis (Fig. 1, 6). The Nocardias showed the widest zones of clearing with the best zone evident in plates seeded with N. brasiliensis. With other systemic fungi, Zymonema (Blastomyc es) dermatitidis and Histoplasma capsulatum, there was definite evidence of retardation of the fungus growth. The results obtained with this crude experiment indicated that better results were to he expected with more specific fractions or extracts especially with the dermatophytes and the Nocardias and to some extent with the systemic fungi but not with the yeastlike organisms. The following experiments were carried out with the various substances, extracts or filtrates as shown in the flow sheet. Crude broth filtrate. This filtrate contained living bacterial cells and as such was consistently producing an active principle. With the small supply of material available, only two organisms were tested, Trichophyton mentagrophytes and Coccidioides immitis. Serial dilutions were made as shown in Table 1. T. ORGANISMS Trichophyton mentagrophytes... Coccidioides immitis TABLE 1 Crude broth filtrate Results read 32 days after inoculation. Trichophyton mentagrophytes was inhibited in dilutions up to 1:50 whereas Coceidioides immitis showed inhibition in dilutions up to 1:250. mentagrophytes showed growth in a dilution of 1:50 and C. immitis in a dilution of 1:250 after 32 days. This experiment was analogous to the use of living organisms. The results coincided roughly with the zones of inhibition produced in the plate cultures but here, a more accurate or quantitative determination could be made of the activity of the antibiotic. Lyophitized crude broth filtrate. This was a lyophilized filtrate of the crude broth and was cell free. Serial dilutions corresponded to those with the crude broth filtrate. The results arc shown in Table 2. Readings were made 20 days after inoculation. T. mentagrophytes and C. immitis were inhibited in a dilution of 1:50 and showed growth in a dilution of 1: 100. The dermatophytes, Microsporum canis, M. gypseum, Epidermophyton fioccosum and Achorion schoenleinii were inhibited in a dilution of 1:100 but grew in a dilution of 1:250. Monilia albicans was unaffected, growing in all dilutions. Cryptococcus neoformans and Sporotrichum schenckii were inhibited through a dilution of 1:20 but grew in 1:50. Zymonema dermatitidis and Histoplasma capsulatum were similar in their response. The best inhibition was exhibited by Nocardia asteroides showing growth in a dilution of 1:500 with no growth in 1:250. Filtrate A. This filtrate represents the original filtered beer. Three organisms Control + +

7 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 271 were used in this experiment because of the small amount of material available. As seen in Table 3, only N. asteroides was inhibited and in a concentration of 1:1,000 with growth in the next dilution 1:10,000. Both C. immitis and T. mentagrophytes were not inhibited. O1GASMS TABLE 2 Lyophilized crude broth filtrate 1:1 1:5 1:10 1:20 1:50 1:100 1:250 1:500 1:750 1:1,000 Microsporum canis Microsporum gypseum Trichophyton mentagrophytes... + Achorion schoenleinii Epidermophyton floccosum Monilia albicans Sporotrichum schenckii Nocardia asteroides Histoplasma capsulatum Cryptococcus neoformans Zymonema dermatitidis Coccidioides immitis Results read 20 days after inoculation. Monilia albicans grew in all dilutions. Sporotrichum schenckii, Histoplasma capsulatum, Cryptococcus neoformans and Zymonema dermatitidis grew in dilutions of 1:50 and greater. Trichophyton mentagrophytes and Coccidioides iinrnitis were inhibited in dilutions up to 1:100. Microsporum canis, M. gypscum, Achorion schoenleinii and Epidermophyton fioccosum were inhibited in a dilution of 1:100 with growth in dilutions of 1:250 and higher. Nocardia asteroides showed best results, being inhibited in dilutions up to 1:500 with growth in dilutions of 1:500 and higher. ORGANISMS Trichophyton mentagrophytes.. Nocardia asteroides Coccidioides immitis TABLE 3 Filtrate A :50 1:100 1:250 1:500 1:1,000 1:10,000 1:100,000 Control Results read 16 days after inoculation. Trichophyton mentagrophytes and Coccidioides immitis were not inhibited in any dilution. Nocardia asteroides was inhibited in dilutions of 1:1,000 with growth in dilutions of 1:10,000 and higher. Filtrate B. This filtrate consists of the so-called spent beer following solvent extraction. In addition to the organisms tested with filtrate A, the fungi Monilia albicans, Sporotrichum schenckii and Zymonema dermatitidis were used. All of the organisms were completely uninhibited with the exception of N. asteroides

8 272 TEE JOURNAL OF INVESTIGATIVE DERMATOLOGY which showed pronounced inhibition in a dilution of 1:10,000 with growth in the next dilution, 1:100,000 appearing after 20 days. It was apparent that the antibiotic became completely spent sometime after the sixteenth day since there was inhibition up until that time in 1:100,000, as seen in Table 4. ORGANISMS TABLE 4 Filtrate B 1:50 1:100 1:250 1:500 1:1,000 1:10,000 1:100,000 Control Trichophyton mentagrophytes Monilia albicans + Sporotrichum schenckii Nocardia asteroides Zymonema dermatitidis Coccidioides immitis Results read 16 and 20 days after inoculation. indicates no growth at 16 days but growth at 20 days. All the organisms with the exception of Nocardia asteroides grew in all the dilutions, some showing delayed growth. N. asteroides showed pronounced inhibition in a dilution of 1:10,000 with delayed growth (after20 days) in a dilution of 1:100,000. ORGANISMS 1:50 TABLE 5 Filtrate C 1:100 Microsporum canis Trichophyton mentagrophytes Nocardia asteroides Cryptococcus neoformans + + Zymonema dermatitidis Coccidioides immitis :250 1:500 1:1,000 1:10,000 1:100,000 Control Results read 17 days after inoculation. Microsporum canis, Trichophyton mentagrophytes and Coccidioides immitis were uninhibited in all dilutions. Cryptococcus neoformans grew in dilutions of 1:500 and higher. Zymonema dermatitidis was inhibited in a dilution of 1:1,000 and Nocardia asteroides in a dilution of 1:10,000. Filtrate C. This filtrate represents the spent solvents. M. canis, T. mentagrophytes and C. immitis were uninhibited. C. neoformans was inhibited in a dilution of 1:250, Z. dermatitidis in a dilution of 1:1,000 and N. asteroides in a dilution of 1:10,000. The results as shown in Table 5 were read 17 days after inoculation. Apparently the fungicidal principle resides in the spent solvents and is not affected by the extraction process. Filtrate D. This filtrate represents the first water extraction from the second

9 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 273 solvent extraction. This filtrate was extracted from filtrate C. As seen in Table 6, T. mentagrophytes and C. immitis were not inhibited whereas N. asteroides was inhibited in a dilution of 1:10,000 after 16 days. ORGANISMS Trichophytonmentagrophytes.. Nocardia asteroides Coccidioides immitis TABLE 6 Filtrate D :50 1:100 1:250 1:500 1:1,000 1:10,000 1:100,000 Control Results read 16 days after inoculation. Trichophyton mentagrophytes and Coccidioides immitis were not inhibited whereas Nocardia asteroides was inhibited through a dilution of 1:10,000. TABLE 7 Filtrate E 1:20 1:40 1:80 1:200 1:1,000 1:10,000 1:100,000 Control ORGANISMS Microsporum canis + + Microsporum gypseum Trichophyton mentagrophytes. + Achorion schoenleinii + Epidermophyton floccosum + Monilia albicans + Sporotrichum schenckii Nocardia asteroides + Nocardiamadurae + Nocardia brasiliensis Histoplasma capsulatum Cryptococcus neoformans Zymonema dermatitidis Results read 16 and 20 days after inoculation. F indicates no growth at 16 days but Coccidioides immitis + growth at 20 days. Microsporum gypseum, Trichophyton mentagrophytes, Monilia albicans, Sporotrichum schenckii and Cryptococcus neoformans were not inhibited. Micro. sporum canis and Coccidioides iminitis grew, after 20 days, in a dilution of 1:40. Achorion schoenleinii grew, after 20 days, in a dilution of 1:80. Epidermophyton floccosum and Histoplasma capsulatum grew, after 16 days, in a dilution of 1:200 whereas Zymonema dermatitidis grew in the same dilution after 20 days. Nocardia asteroides and N. madurae grew, after 20 days, in a dilution of 1:100,000 but N. braciliensis was not inhibited in 1:100,000. Filtrate E. This filtrate is composed of the water extract of the spent solvents (filtrate C) from the bacitracin recovery process. Twelve organisms were used in this experiment as listed in Table 7. The results were read 16 and 20 days following inoculation. M. gypseum, T. mentagrophytes, M. albicans, S. schenckii

10 274 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY and C. neoformans were not inhibited. M. canis and C. immitis were inhibited in a dilution of 1:40 after 16 days with growth apparent in this dilution after 20 days. A. schoenleinii was inhibited in a dilution of 1:80 after 16 days with growth evident in this dilution after 20 days. E. fioccosum, H. capsulatum and Z. dermatitidis were inhibited in a dilution of 1:80 with growth in the next dilution 1: 200. With Z. dermatitidis growth occurred in this dilution after 20 days. The actinomycetes, N. asteroides, N. madurae and N. brasiliensis showed inhibition in dilutions up to and including 1:100,000. N. asteroides and N. madurae, however, showed slight growth in this dilution after 20 days. TABLE 8 Filtrate F DILUTZONS O2OANXSMS C Trichophyton mentagrophytes Moniliaalbicans Sporotrichum schenckii Nocardia asteroides.... Nocardia madurae Nocardia brasiliensis... Zymonema dermatitidis. Coccidioides immitis C Control. Results read 14 days after inoculation. Trichophyton mentagrophytes, Monilia albicans, Sporotrichum schenckii, Zymonema dermatitidis and Coccidioides immitis were not inhibited in any dilution. Nocardia asteroides and N. Madurae showed inhibition in a dilution of 1:10,000 with growth in 1:50,000. N. brasiliensis showedinhibition inl:750,000 with growth in a dilution of 1:1,000,000. Filtrate F. This filtrate is bacitracin. Eight organisms were employed as seen in Table 8. The species specificity of this extract was well demonstrated by the fact that T. mentagrophytes, M. albicans, S. schenckii, Z. dermatitidis and C. inunitis were unaffected whereas N. asteroides and N. madurae were inhibited by a dilution of 1:10,000 with growth in a dilution of 1: 50,000. N. brasiliensis was inhibited in a dilution of 1:750,000 with growth occurring in a dilution of 1:1,000,000. Readings were made after 14 days. Filtrate G. This filtrate represents the solvents following the second extraction. Five organisms were utilized as listed in Table 9. Again, T. mentagrophytes and C. immitis were uninhibited. N. asteroides and N. madurae were inhibited in dilutions of 1:10,000 with growth in a dilution of 1:25,000. N. brasiliensis again showed the best inhibition with no growth in a dilution of 1:250,000 and growth in a dilution of 1:500,000. The results were read 14 and 17 days after inoculation.

11 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 275 Filtrate H. This filtrate represents the material following the second extraction as found in the water layer. The same organisms were used here as with filtrate G. Final readings were made 17 days following inoculation. T. men- ORGANISMS TABLE 9 Filtrate G r g. Trichophyton mentagrophytes Nocardia asteroides Nocardia madurae Nocardia brasiliensis Coccidioides immitis Results read 14 days after inoculation. Trichophyton mentagrophytes and Coccidioides immitis were uninhibited. Nocardia asteroides and N. xnadurae were inhibited in dilutions of 1:10,000 with growth in a dilution of 1:25,000. N. brasiliensis grew in a dilution of 1:500,- 000 with inhibition in 1:250,000. TABLE 10 Filtrate H ORGAOSMS o 8-9 ',c a 9 0 9e 9 ' X, 9 - Trichophyton mentagrophytes Nocardia asteroides... + Nocardia madurae Nocardia brasiliensis... Coccidioides immitis :10 1:1,000,000. 1:1.5 X 10 = 1:1,500,000. C Control. Results read 17 days after inoculation. Trichophyton mentagrophytes and Coccidioides immitis showed inhibition in a dilution of 1:250 with growth in 1:500. Nocardia asteroides and N. madurae were inhibited in a dilution of 1:10,000 with growth in 1:25,000. N. brasiliensis was inhibited in a dilution of 1:1,000,000 with growth in 1:1,500,000. tagrophytes and C. immitis were inhibited in a dilution of 1:250 with growth occurring in a dilution of 1: 500. N. asteroides and N. madurae were inhibited in a dilution of 1:10,000 with growth occurring in a dilution of 1:25,000. N. brasiliensis showed inhibition in a dilution of 1:4,000,000 for 11 days but showed inhibition only through a 1:1,000,000 dilution after 14 days where it remained

12 276 THE JOURNAL OF INVESTIGATiVE DERMATOLOGY after 17 days. There is a definite specificity for N. brasiliensis with this filtrate. The results are shown in Table 10. Additional experiments. In addition to testing the various filtrates, an experiment was set up to include bacitracin in the medium in various concentrations with the addition of 50 per cent human serum. The single organism N. asteroides was used. Readings made 14 days after inoculation showed inhibition in a dilution of 1:10,000 with growth in the next dilution (1: 25,000). Apparently, the serum did not exert any influence on the inhibition or growth of the fungus. TABLE 11 Bacitracin renewal experiment OROANI5ISS Nocardia asteroides Nocardia madurae Nocardia brasiliensis 15 days 15 days 15 days 1:10,000 1:25,000 1:50,000 1:75,000 1:100,000 1:200, :500, :750, :1,000, :1,500, :2,000, :2,500, :3,000, :4,000, :5,000, Control per cent of original concentration of bacitracin added on fifth and tenth day following inoculation. Results read 15 days following inoculation. Nocardia asteroides showed inhibition in a dilution of 1:100,000. N. madurae was inhibited in a dilution of 1:1,000,000 with growth in 1:1,500,000. N. brasiliensis was inhibited in a dilution of 1:2,500,000 with growth in 1:3,000,000. A second additional experiment consisted of renewing the bacitracin in the medium on the fifth and tenth day following inoculation. The bacitracin was originally added to the medium in varying concentrations. On the fifth and tenth day respectively, 25 per cent of the original concentration was added on the basis that bacitracin loses that much of its potency on standing in an aqueous solution. Readings were made on the fifteenth day following inoculation. The three Nocardias were employed. N. asteroides was inhibited in a dilution of 1:100,000 with growth in a dilution of 1:200,000. N. madurae was inhibited in a dilution of 1:1,000,000 with growth in a dilution of 1:1,500,000. N. brasiliensis was inhibited in a dilution of 1:2,500,000 with growth occurring in a dilution of 1:3,000,000. The results when compared with those of the other experiments,

13 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 277 including the various filtrates of the bacitracin recovery process and with bacitracin itself, support the contention that bacitracin is weakened in an aqueous solution and that renewal of the antibiotic is essential for it to exert its optimum antibiotic activity. DISCUSSION It is apparent from the literature that various strains of Bacillus subtilis extracted by different methods, may give rise to antibiotics with a varied antifungal spectrum. A review of the several reported antibiotics with such capabilities clearly demonstrates this. Katznelson (4) used chiefly plant pathogens and a few aerobic actinomycetes. He found that Nocardia asteroides grew poorly in a concentration of 60 per cent by volume and not at all in a concentration of 80 per cent by volume. Salle and Jann (8) worked with subtilin and found several Nocardias, including N. asteroides, to be inhibited by a concentration of 1: 1,000 whereas nonsusceptible organisms included Monilia albicans, Cryptococcus neoformans, Sporotrichum schenckii and Trichophyton mentagrophytes. Lewis, Hopper and Shultz (5) incorporated culture filtrates in medium and were able to inhibit T. mentagrophytes in a dilution of 1:100. Microsporum gypseum was inhibited in a dilution of 1:20, Monilia alibicans in a dilution of 1:10 and Sporotrichum schenckii in a dilution of 1:20. Johnson and Burdon (11) found that eumycin in a concentration of 0.1 to 0.3 mgm. (dry weight) per ml. of medium prevented entirely or definitely inhibited the growth of Trichophyton mentagrophytes, Microsporum gypseum, Epidermophyton fioccosum and related species of fungi, including Nocardia. The effect of eumycin was slightly less on Sporotrichum schenckii and Hormodendrum and it had no effect on Monilia albicans or Cryptococcus neoformans. The best antifungal spectrum with a Bacillus subtilis antibiotic was that obtained by Landy, Warren, Rosenman and Colio who employed bacillomycin. The dermatophytes including Microsporum audouini, M. gypseum, Trichophyton mentagrophytes, T. rubrum, Epidermophyton fioccosum and Achorion schoenleinii were inhibited in a dilution of mgm. bacillomycin per ml. of medium or a dilution of 1:75,000. Of the more invasive fungi, Coccidioides immitis, Histoplasma capsulatum, Monosporium apiospermum and Paracoccidioides braziliensis were also inhibited in the same concentration (0.025 mgm. bacillomycin per ml. medium). The mycelial form of Zymonema dermatitidis was inhibited in a concentration of mgm. bacillomycin per ml. medium or 1:100,000 dilution, whereas the yeast form of the fungus was more resistant being inhibited in a dilution of mgm. bacillomycin per ml. medium or a dilution of 1:750,000. Monilia albicans, Cryptococcus neoformans and Sporotrichum schenckii were inhibited in a dilution of 0.05 mgm. bacillomycin per ml. medium or a dilution of 1:50,000. On the other hand, Phialophora verrucosa, P. pedrosoi and Nocardia asteroides were not inhibited in a dilution as low as 0.5 mgm. bacillomycin per ml. medium or a dilution of 1:5,000. In sharp contrast to bacillomycin which had no effect on Nocardia, bacitracin,

14 278 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY the antibiotic obtained from the Tracy strain of Bacillus subtilis shows a strongly inhibitory action on Nocardia asteroides, N. madurae and N. brasiliensis (Table 8). The former two are inhibited in a dilution of 1:10,000 while the latter is prevented from growing in a dilution of 1: 750,000. The fungi T. mentagrophytes, M. albicans, S. schenckii, Z. dermatitidis and C. immitis, again in sharp contrast are not inhibited by bacitracin in a dilution as low as 1:50. It is apparent that the antifungal property of this strain of Bacillus subtilis is lost or destroyed with some of the filtrates and altered with others as compared with the antifungal activity elicited when the crude filtrates with or without living organism are used. In filtrates A, B, C, D, F (bacitracin) and G, the antibiotic activity is limited to the Nocardias with only Z. dermatitidis being inhibited in a dilution of 1:1,000 by filtrate C whereas in filtrates E and H there is some activity against other pathogenic fungi, with the best results being obtained with filtrate H. T. mentagrophytes and C. immitis are inhibited in a dilution of 1:250 by filtrate H. The inhibition of the aerobic Actinomycetes (Nocardias) varies with the different filtrates. N. asteroides is inhibited by filtrate A in a dilution of 1: 1,000 and in a dilution of 1:10,000 by filtrates B, C, D, E, F, G and H. N. madurae likewise is inhibited in a dilution of 1:10,000 by filtrates E, F, G and H. N. brasiliensis on the other hand appears to be most susceptible to the various filtrates being inhibited in a dilution of 1:100,000 by filtrate E, 1: 750,000 by filtrate F, 1:250,000 by filtrate G and 1: 1,000,000 by filtrate H. In comparing the three Nocardias it would appear that N. braziliensis is a very unstable organism as contrasted with the stable inhibitory responses of N. asteroides and N. madurae to the various filtrates. In the presence of human serum (50 per cent in Sabouraud's broth), N. asteroides was inhibited in a dilution of 1:10,000 bacitracin. The serum apparently had no effect on the bacitracin. The best results obviously were obtained with the bacitracin renewal experiment. Twenty-five per cent of the original concentration of bacitracin was added on the fifth and tenth day following inoculation. The results read on the fifteenth day following inoculation showed that N. asteroides was inhibited in a dilution of 1:100,000, ten times the inhibition found in the various filtrates. N. madurae was inhibited in a dilution of 1:1,000,000, one hundred times the inhibition found in the filtrates. N. brasiliensis was inhibited in a dilution of 1:2,500,000 which is considerably greater than that obtained with the filtrates. It may be concluded from the evidence in the literature and from the results obtained here that in the field of medical mycology, the antibiotics derived from Bacillus subtilis are chiefly effective against the aerobic Actinomycetes. Two antibiotics, however, that obtained from the Marburg strain and named eumycin and that named bacillomycin, show activity against other fungi. In sharp contrast, bacillomycin is strongly active against many of the pathogenic fungi but is relatively inactive against the Nocardias. Bacitracin is strongly active against the Nocardias but relatively inactive against other pathogenic fungi.

15 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS 279 Evidence is accumulating, as a result of the examination of material from numerous pulmonary cases suspected of being tuberculosis, that mycoses due to Nocardia asteroides are more frequent than is commonly suspected. In the past few months, five cases with lung disease and one with endocarditis, all caused by N. asteroides as proven by cultures, have been so diagnosed by the senior author. In such cases the use of bacitracin could have been of great value. Unfortunately, bacitracin is not yet available commercially for injection purposes. The antibiotic, however, has been tested experimentally in humans and it has been found that intramuscular injections of bacitracin may be given in doses of 10,000 units or larger every four to six hours. There may be a local reaction at the site of injection which can be lessened by combining procaine or novocaine with the injected antibiotic. Patients who have received newer preparations of bacitracin by injection over a long period of time have exhibited minimal toxic symptoms. These patients had to be watched particularly for albuminuria. Some patients with bacterial infections resistant to penicillin and streptomycin have tolerated large amounts of bacitracin by injection with beneficial results. Work is progressing at the present time on the preparation of a purer and less toxic supply of bacitracin. SUM?VIABY In an attempt to find an antibiotic active against pathogenic fungi, various filtrates of the Bacillus subtilis (Tracy strain) were employed. These consisted of whole cultures (living cells), crude broth filtrates (live cells), lyophilized crude broth filtrate (cell free) and the several filtrates obtained in the bacitracin recovery process. The crude filtrates, including the whole culture, exerted an inhibitory action on the fungi used. This was not apparent in dilutions greater than 1:250 with the crude filtrates, either with or without living cells. Some of the fungi were inhibited in dilutions no greater than 1:20, others in dilutions no greater than 1:50 or 1:100. Various filtrates developed in the bacitracin recovery process were employed in order to determine whether such filtrates might possibly contain antifungal properties. These consisted of eight filtrates labelled in the order in which they were formed in the recovery process. Filtrates A and B exerted an action only on Nocardia asteroides in dilutions of 1: 1,000 and 1: 10,000 respectively. Filtrate o had antifungal properties for Cryptococcus neoformans (1:250), Zymonema dermatitidis (1:1,000) and Nocardia asteroides (1:10,000). Filtrate D also affected Nocardia asteroides (1:10,000 dilution). Filtrate E had mild antibiotic activity for Microsporum canis (1:20), Achorion schoenleinii (1:40), Epidermophyton fioccosum (1:80), Histoplasma capsulatum (1:80), Zymonema dermatitidis (1:80) and Coccidioides imxnitis (1:20). Antibiotic activity was manifest with the Nocardias: N. asteroides (1:10,000; N. madurae (1: 10,000); and N. brasiliensis (1:100,000). Filtrate F showed antibiotic activity only with the Nocardias: N. asteroides (1:10,000); N. madurae (1:10,000); and N. brasiliensis (1:750,000). Filtrate G likewise showed activity only against the Nocardias:

16 280 THE JOIJRNAL OF INVESTIGATIVE DERMATOLOGY N. asteroides (1: 10,000); N. madurae (1: 10,000); N. brasiliensis (1: 250,000). Filtrate H showed mild activity against Trichophyton mentagrophytes (1:250), Coccidioides immitis (1:250) but strong activity against the Nocardias: N. asteroides (1:10,000); N. madurae (1:10,000); and N. brasiliensis (1: 1,000,000). With all the filtrates the activity against N. asteroides and N. madurae remained constant (1:10,000) whereas against N. brasiliensis the activity ranged from 1:100,000 through 1: 1,000,000. The addition of 50 per cent human serum to the test medium did not alter the activity of bacitracin against N. asteroides. The best results were obtained by the renewal of the bacitracin in the amount of 25 per cent of the original concentration added to the medium on the fifth and tenth day following inoculation. After 15 days, N. asteroides was inhibited in a dilution of 1:100,000, N. madurae in a dilution of 1:1,000,000 and N. brasiliensis in a dilution of 1: 2,500,000. Bacitracin and the various filtrates produced in the recovery process, therefore, are mildly active to inactive against most pathogenic fungi and strongly active against the Nocardias. REFERENCES 1. WAXSMAN, S. A.: Antagonistic relations of microorganisms. Bact. Revs. 5: (Sept.) WEIDMAN, F. D.: Laboratory aspects of epidermophytosis. Arch. IDermat. & Syph. 15: (April) CHAMBERS, S., AND WEIDMAN, F. D.: A fungistatic strain of Bacillus subtilis isolated from normal toes. Arch. Dermat. & Syph. 18: (Oct.) KATZNELSON, H.: Inhibition of microorganisms by a toxic substance produced by an aerobic spore-forming bacillus. Canadian J. Res., Sect. C, 20: 169 (March) LEwis, G. M., HOPPER, M. E., AND SHULTZ, S.: In vitro fungistasis by a bacterium (Bacillus subtilis var. XG and XY). Arch. Dermat. & Syph. 54: (Sept.) BENEDICT, R. G., AND LANGLYKKE, A. F.: Antibiotics. Ann. Rev. Microbiol. 1: , JANSEN, E. F., AND HIRSCHMANN, D. J.: Subtilin An antibacterial product of Bacillus subtilis. Culturing conditions and properties. Arch. Biochem. 4: (July) SALLE, A. J., AND JANN, G. J.: Subtilin An antibiotic produced by Bacillus subtilis. I. Action on various organisms. Proc. Soc. Exper. Biol. & Med. 60: 60 64, JOHNSON, B. A., ANKER, N., AND MELENEY, F. L.: Bacitracin; A new antibiotic produced by a member of the B. subtilis group. Science 102: 376 (Oct. 12) FOSTER, J. W., AND WOODRUFF, H. B.: Bacillin, a new antibiotic substance from a soil isolate of Bacillus subtilis. J. Bact. 51: , JOHNSON, E. A., AND BURDON, K. L.: Eumycin A new antibiotic active against pathogenic fungi and higher bacteria, including bacilli of tuberculosis and diphtheria. J. Bact. 51: 591, VALLEE, M.: Etude du pouvoir Bactériolytique de Bacillus subtilis. Compt. rend. Soc. Biol. 139: , SAINT-RAT, L. DR, ET OLIvIER, H. R.: Extraction et purification de L'endosubtilysine. Compt. rend. 222: , RAMON, G., ET Ricriou, R.: De l'infiuence sur les propriétés antibiotiques et antidotiques des filtrats de culture du B. subtilis, de Ia chaleur et de l'aldehyde formique agissant simultanément. Compt. rend. 222: , 1946.

17 PROPERTIES OF EXTRACTS OF BACILLUS SUBTILIS CALLOW, R. K., AND D'ARcr HART, P.: Antibiotic material from Bacillus licheniformis (Weigmann emend. Gibson) active against species of Mycobacteria. Nature 157: , LANDY, M., ROSENMAN, S. B., AND WARREN, G. H.: An antibiotic from Bacillus subtilis active against pathogenic fungi. J. Bact. 54: 24 (July) LANDY, M., WARREN, G. H., ROSENMAN, S. B., AND CoLlo, L. G.: Bacillomycin: An antibiotic from Bacillus subtilis active against pathogenic fungi. Proc. Soc. Exper. Biol. & Med. 67: (April) HIRSOHHORN, H. N., BUCCA, M. A., AND THAYER, J. D,: Subtenolin: An antibiotic from Bacillus subtilis. I. Bacteriologic properties. Proc. Soc. Exper. Biol. & Med. 67: (April) HOWELL, S. F., AND TAURER, H.: Subtenolin. An antibiotic from Bacillus subtilis. II. Isolation and chemical properties. Proc. Soc. Exper. Biol. & Med. 67: (April) 1948.